Powerful Antioxidants and Cytotoxic Activities of the Methanol Extracts from Eight Soybean Cultivars

In the present study, the chemical composition and total phenolic (TPC) and total flavonoid contents (TFC) of eight soybean cultivars (Giza 21, Giza 22, Giza 35, Giza 111, Giza 82, Giza 83, Crawford, and Holliday) were estimated. Moreover, antioxidant activity and in vitro cytotoxic activities against HepG-2 and MCF-7 were evaluated. Giza 21, Giza 111, and Crawford cultivars recorded higher than 40% crude protein. The analysis revealed that TPC values in seed extracts ranged from 10.5 mg GAE/g extract in Giza 35 to 6.4 mg GAE/g extract in Giza 22. TFC varied from 1.20 mg QE/g extract in Giza 111 to 0.55 mg QE/g extract in Crawford. Giza 35 exhibited the highest content of genistein and daidzein and the highest free radical scavenging activity (61.833%). The results of the MTT assay demonstrated that the soybean methanolic extracts inhibited the proliferation of HepG-2 and MCF-7 cells in a dose-dependent manner. Giza 35 exhibited the highest cytotoxic activity. In conclusion, Giza 35 cultivar recorded the highest TPC and TFC values and antioxidant and cytotoxic activities. Therefore, this cultivar can be used as a source for the production of pharmaceutical and medicinal products rather than as a nutritional source of protein.


Introduction
Natural bioactive compounds isolated from different sources have protective activities against various diseases such as cancer [1]. Among these natural components, polyphenols have received special attention due to their potent antibacterial, antiviral, antioxidant activities and because they inhibit the proliferation of cancer cells [2]. Due to their biological activities, polyphenols have been extensively researched for decades. Consequently, polyphenols isolated from different agricultural crops have been investigated with the aim of supplying nutrition that has useful effects on human health [3].
Among these agricultural crops, legume seeds have received special attention due to their natural bioactive components, such as phenolics, flavonoids, and isoflavones [4].
Soybean (Glycine max (L.) Merr.) is one of the world's most widely planted leguminous crops, growing in tropical, subtropical, and temperate climates and supplying plentiful protein (about 40%) and oil (about 20%) for human and animal consumption [5]. Many of the phenolic compounds present in plant tissues have antioxidant properties. All phenolics have reactive oxygen species (ROS) scavenger capabilities. Most phenolic compounds are concentrated in the seed of the legumes [6]. The bioactive phenolic compounds present in grain legumes make them suitable candidates for creating new functional foods [7]. These compounds might offer indirect protection by activating endogenous defense systems and by the modulation of cellular signaling processes. The change in the bioactivity of phenolic compounds exemplifies their importance in food products. They have many health benefits, such as anticarcinogenic, antioxidant, and antimicrobial properties [8,9]. Except for isoflavonoids, limited research has been conducted on the other phenolic classes present in soybean.
The local soybean varieties have a wide range of maturity and diverse morphology. Apart from these, they are high-yielding with good desirable agronomic characteristics under intercropping conditions. Thus, this study aims to examine the chemical composition of 6 local soybean cultivars ( Giza 21,22,35,82,83, and 111) compared with American cultivars of soybean such as Crawford and Holliday. On the other hand, seed phenolic-rich extracts of different eight soybean cultivars as antioxidant and cytotoxic agents were estimated.

Proximate Analysis
Descriptive values of eight soybean cultivars for proximate analysis (crude protein, ash, fat, crude fiber, carbohydrate, and moisture contents), total phenolic (TPC), and total flavonoid contents (TFC) are presented in Table 1. Crude protein values ranged between 35.17% in Giza 35 and 44.23% in Giza 111. Cultivars recorded higher than 40% crude protein were Giza 21, Giza 111, and Crawford. Moisture content ranged from 3.57% in Holliday to 5.20% in Giza 111. Ash content in eight soybean cultivars ranged from 4.05% in Giza 21 to 5.16% in Holliday. Total fat also varied from 20.97% in Giza 35 to 18.15% in Giza 82. The carbohydrate content ranged from 27.70% in Holliday to 20.49% in Giza 111. The crude fiber content varied from 10.85% in Giza 35 to 6.14% in Crawford. The results revealed nonsignificant differences among some cultivars and significant differences among others in constituents, reflecting the variations in genetic background and/or origin. All data are expressed as the mean (n = 3) with ±SD in the same column; the a-f letters indicate significant differences between means (p < 0.01).

Total Phenolic (TPC) and Total Flavonoid Content (TFC)
TPC (mg GAE/g extract) and TFC (mg QE/g extract) of extracts from soybean cultivars are presented in Table 2. A wide variation was observed for the TPC and TFC and the cultivars differed significantly with respect to these parameters. The analysis revealed that TPC values in seed extracts range from 10.5 mg GAE/g extract in Giza 35 to 6.4 mg GAE/g extract in Giza 22. TFC varied from 1.20 mg QE/g extract in Giza 111 to 0.55 mg QE/g extract in Crawford. All data are expressed as the mean (n = 3) with ±SD in the same column; the a, b, c, d and e letters indicate significant differences between means (p < 0.01).

Phenolic, Flavonoid, and Isoflavone Compounds Identification by HPLC
A representative chromatogram of the HPLC phenolic compounds analysis of eight soybean cultivars extracts is shown in Figure 1. Nine peaks (syringic, quercetin, gallic acid, benzoic acid, genistein, daidzein, p-coumaric, glycitein, and ferulic acid) dominated in the chromatograms from the separation of Giza 21, Giza 22, Giza 35, Giza 82, Giza 83, and Holliday extracts. On the other hand, benzoic acid and ferulic acid were absent on chromatograms from the separation of Giza 111 and Crawford, respectively. Table 3 summarizes the contents of phenolic compounds 1-9 in the eight soybean cultivars extracts. It was shown that quercetin, genistein, daidzein, and glycitein were the main phenolic compounds in the eight soybean cultivar extracts. The main flavonoid compound of the eight soybean cultivars was quercetin. Giza 111 exhibited the highest content of quercetin (37.34%), followed by Giza 35 (30.80%), whereas Giza 83 recorded the lowest quercetin amount (26.05%). An HPLC chromatogram shows the isoflavone compounds (genistein, daidzein, and glycitein). Giza 35 exhibited the highest content of genistein and daidzein (30.00% and 15.07%, respectively), whereas Giza 111 recorded the lowest genistein and daidzein values (15.87% and 1.87%, respectively). The main phenolic compound of the eight soybean cultivars was gallic acid. The highest gallic acid content was recorded in the case of Giza 35 (7.79%), followed by Giza 111 and Giza 83 (7.53% and 7.50%, respectively). All data are expressed as the mean (n = 3) with ±SD in the same column; the a, b, c, d and e letters indicate significant differences between means (p < 0.01).

Antioxidant Activity
The antioxidant activities of the eight soybean cultivars are shown in Table 4. Giza 35 exhibited the highest free radical scavenging activity (61.833%), whereas Giza 82 had the lowest (47.928%). There were significant differences (p < 0.001) in the free radical scavenging activities of the Giza 35 and others cultivars (data not shown). The extracts from all soybean cultivars demonstrated the capability to scavenge DPPH free radicals. Overall, antioxidant activity increased proportionally to the increased concentration, and a linear relationship between DPPH radical scavenging activity and concentration was established (data not shown). The results of the IC50 value of the soybean cultivar methanolic extract are presented in Table 5. Giza 35, Giza 111, Giza 83, Giza 21, and Crawford methanol extracts had stronger antioxidant activities than TBHQ. The required concentrations for scavenging of 50% radicals were 45, 52, 55, 57, 61, and 62 mg/mL for Giza 35, Giza 111, Giza 83, Giza 21, Crawford, and TBHQ, respectively. <0.001 All data are expressed as the mean (n = 3) with ±SD in the same column; the a, b, c, d, e and f letters indicate significant differences between means (p < 0.01).

Antioxidant Activity
The antioxidant activities of the eight soybean cultivars are shown in Table 4. Giza 35 exhibited the highest free radical scavenging activity (61.833%), whereas Giza 82 had the lowest (47.928%). There were significant differences (p < 0.001) in the free radical scavenging activities of the Giza 35 and others cultivars (data not shown). The extracts from all soybean cultivars demonstrated the capability to scavenge DPPH free radicals. Overall, antioxidant activity increased proportionally to the increased concentration, and a linear relationship between DPPH radical scavenging activity and concentration was established (data not shown). The results of the IC 50 value of the soybean cultivar methanolic extract are presented in Table 5. Giza 35, Giza 111, Giza 83, Giza 21, and Crawford methanol extracts had stronger antioxidant activities than TBHQ. The required concentrations for scavenging of 50% radicals were 45, 52, 55, 57, 61, and 62 mg/mL for Giza 35, Giza 111, Giza 83, Giza 21, Crawford, and TBHQ, respectively. All data are expressed as the mean (n = 3) with ±SD in the same column; the a, b, c, d, e and f letters indicate significant differences between means (p < 0.01). All data are expressed as the mean (n = 3) with ±SD in the same column; the a, b, c and d letters indicate significant differences between means (p < 0.01). Table 5 presents the antioxidant activity of soybean cultivars methanolic extract at 800 µg/mL, determined as FRAP. FRAP test showed significant differences between Giza 35 and other soybean cultivars. FRAP values ranged from 770 to 1900 µmol Fe +2 /g extract. FRAP was the highest for Giza 35 (1900 µmol Fe +2 /g extract), followed by those of Giza 83 (1600 µmol Fe +2 /g extract) and Giza 111 (1400 µmol Fe +2 /g extract). No significant differences were observed (p < 0.001) between Giza 111 and Giza 83. FRAP was the lowest for Holliday (770 µmol Fe +2 /g extract), with no significant differences (p < 0.001) between Giza 21, Giza 22, and Holliday.

Cytotoxic Activity
Soybean methanolic extracts at different concentrations (12.5, 25, 50, 100, and 200 µg/mL) from eight soybean cultivars were tested in vitro for their cytotoxic activities on the HepG-2 and MCF-7 human cancer cell lines using the MTT assay. The percentages of the viable cells and their cytotoxic activity were measured and compared with the control, Doxorubicin (Tables 6 and 7). Overall, cell viability (%) decreased with the increase in concentration, and a linear relationship between cell viability and concentration was established. Cytotoxic activity of methanolic extracted from soybean Giza 35 (the highest cytotoxic activity) at different concentrations (12.5, 25, 50, 100, and 200 µg/mL) against HepG-2 (A) and MCF-7 (B) was compared to that of Doxorubicin as a control (Figure 2). The results of the MTT assay demonstrated that the soybean methanolic extracts inhibited the proliferation of HepG-2 and MCF-7 cells in a dose-dependent manner. All data are expressed as the mean (n = 3) with ±SD in the same column; the a-r letters indicate significant differences between means (p < 0.01). All data are expressed as the mean (n = 3) with ±SD in the same column; the a-v letters indicate significant differences between means (p < 0.01).

Discussion
The main objectives of the current study were to investigate the chemical composition of eight soybean cultivars (Giza 21, Giza 22, Giza 35, Giza 111, Giza 82, Giza 83, Crawford, and Holliday), TPC, TFC, antioxidant activity and cytotoxic activity against human  All data are expressed as the mean (n = 3) with ±SD in the same column; the a-r letters indicate significant differences between means (p < 0.01). All data are expressed as the mean (n = 3) with ±SD in the same column; the a-v letters indicate significant differences between means (p < 0.01).

Discussion
The main objectives of the current study were to investigate the chemical composition of eight soybean cultivars (Giza 21, Giza 22, Giza 35, Giza 111, Giza 82, Giza 83, Crawford, and Holliday), TPC, TFC, antioxidant activity and cytotoxic activity against human cell lines (HepG-2 and MCF-7). The results of chemical composition revealed no significant differences among some cultivars and significant differences among others in constituents, reflecting the variations in genetic background and/or origin. The values observed in the present study were similar to those obtained by other researchers [10,11].
In the present study, methanolic extracts from eight soybean cultivars were prepared and analyzed for TPC and TFC. Giza 35 exhibited the highest TPC and TFC and recorded the highest genistein, daidzein, and quercetin contents. Overall, when TPC and TFC are increased in soybean samples, the antioxidant activity increases, as indicated by the results of DPPH and FRAP. The antioxidant activity of the phenolic-rich cultivar (Giza 35) showed high levels of DPPH radical scavenging activity. Furthermore, the values observed in the present study were in line with those obtained by other researchers [12][13][14].
The Folin-Ciocalteu method responds differently to different phenolic chemicals in food matrix samples depending on the number of phenolic groups they contain [15]. Because of the phenolic moiety's reactivity, some polyphenolic compounds have been found to have antioxidant properties, scavenging free radicals via electron donation or hydrogen donation [16]. Isoflavones make up the majority of TPC in soybean seeds. According to several studies, TPC and total isoflavone concentration are favorably associated [17][18][19]. In the present work, HPLC was used to identify phenolic compounds. Nine peaks (syringic, quercetin, gallic acid, benzoic acid, genistein, daidzein, p-coumaric acid, glycitein, and ferulic acid) dominated different soybean cultivars. Previous studies have reported that soybean contains several phenolic acids, such as syringic, ferulic, sinapic, p-coumaric, hydroxybenzoic, caffeic, and chlorogenic acids, and those total phenolic compounds are highly positively correlated with hydroxybenzoic acids, such as gentisic acid and salicylic acid [20].
In comparison to traditional medicines, dietary phytochemicals have been shown to prevent disease start or progression with far less damage and cost [21]. Phytochemicals' efficiency in the therapy of many types of cancer has been extensively studied [22][23][24]. On this therapeutic basis, natural compounds regulate different cellular pathways and enzyme activities that are often altered in cancer cells.
In the current work, soybean methanolic extracts from eight soybean cultivars were tested in vitro for their cytotoxic activities compared with the control, Doxorubicin, on the HepG-2 and MCF-7 human cancer cell lines using the MTT assay. The results of the MTT assay demonstrated that the soybean methanolic extracts inhibited the proliferation of HepG-2 and MCF-7 cells in a dose-dependent manner. Giza 35 recorded the highest cytotoxic activity compared to other cultivars and exhibited the highest TPC and TFC. Furthermore, it recorded the highest genistein, daidzein, and quercetin contents. Overall, the trend in cytotoxic activity was similar to that of TPC, TFC, and isoflavones content. Flavonoids represent an important group of bioactive compounds derived from soybean with known biological activity in cells. From the modulation of inflammation to the inhibition of cell proliferation, flavonoids have been described as important therapeutic adjuvants against several diseases, including diabetes, arteriosclerosis, neurological disorders, and cancer [25]. Various studies have suggested different mechanisms for anticancer action of phenolic compounds or polyphenol-rich extracts. Insulin-like growth factor (IGF), known as a potential biomarker, plays a key role in the development of many cancers, including breast, lung, and colorectal cancers. It is thought that the high activity of these multifunctional peptides (IGFs) plays a meaningful role in all stages of carcinogenesis. Therefore, finding potential inhibitors of IGF should be a primary focus of research. Polyphenols might be promising inhibitors, as they do not have side effects when used in chemoprevention and cancer therapy [26]. Polyphenols exhibit cytotoxicity effects against numerous kinds of malignancies, including colorectal, multiple myeloma, breast, pancreatic, prostate, oral, and lung cancers, mainly described in previously published literature. The cytotoxicity effect of polyphenolic compounds may be related to induced apoptosis by cell cycle arrest at the G2/M phase and rapid accumulation in the cell [27]. Plant polyphenols can prevent cancer development by modulating some of the signal transduction pathways related to the cancer process. The main anticancer mechanisms include modulation of proinflammatory cytokines and modulation of several apoptotic proteins like NF-kB, cyclooxygenase-2, STAT3, and endothelin-1 [28].

Proximate Analysis
Soybean samples were analyzed in triplicate for crude proteins, moisture, total ash, fat, crude fiber, and carbohydrate using the procedures given in the AOAC [29]. All proximate values were provided in g/100 g dry weight [30].

Phenolic Compounds Extraction and Characterization
Seeds from eight cultivars of soybeans were ground and defatted for 6-8 h using a Soxhlet apparatus with hexane. Phenolic compounds were extracted from the defatted raw seeds with 70% (v/v) aqueous methanol at a solid-to-solvent ratio of 1:10 (w/v) at 40 • C min in a Soxhlet apparatus. The extracts were filtered through a filter paper and concentrated in a rotary evaporator (Büchi rotary evaporator, Flawil, Switzerland) below 40 • C. The resultant aqueous solutions were frozen and lyophilized [31].

Total Flavonoid Content (TFC) Estimation
TFC in the soybean seeds' methanolic extracts (2000 µg/mL) was estimated according to the protocol of Ordonez et al. [32] as described in Abdel-Shafi et al. [8] Quercetin (10-1000 µg/mL) was used to obtain the following calibration equation: y = 0.0012x + 0.008; R 2 = 0.944, where y and x are the quercetin absorbance and concentration in µg/mL, respectively. The absorbance of color was recorded at 420 nm by using a spectrophotometer (Jenway, 6405 UV/Vis, Chelmsford, UK).

Polyphenolic Compounds Identification
High-Performance Liquid Chromatography (HPLC) was used to identify polyphenolic compounds in the methanolic extracts of eight soybean seeds cultivars. HPLC-Agilent 1100 apparatus is composed of a C18 column (125 mm × 4.60 mm, 5 µm particle size), two LC pumps, and a UV/Vis detector. The phenolic acid conditions used were as follows: column temperature, 30 • C; injection volume, 20 µL; wavelength, 280 nm; total HPLC run time, 50 min; a mobile phase solution A of 1% aqueous solution acetic acid and solution B of acetonitrile (34,35). The mobile phase was programmed as follows: 0-5 min, solution B at 5-15%; 5-35 min, solution B at 15-35%; 35-40 min, solution B at 35-45%; 40-50 min, solution B at 45-5%. The mobile phase was pumped at a constant flow rate of 1.0 mL/min. The isoflavone conditions were as follows: column temperature, 20 • C; injection volume, 20 µL; wavelength, 260 nm; total HPLC run time, 50 min; mobile phase solution A of 0.1% aqueous solution acetic acid and solution B of acetonitrile. The mobile phase was identical to the one used for the method for the phenolic acids. Before analysis, all samples were filtered through a 0.45 µm membrane filter (Millipore, Billerica, MA, USA). Samples were quantified by comparing the retention times with known authentic standards. All measurements were done in triplicate.

Antioxidant Capacity (DPPH Assay)
The scavenging effect of phenolic compounds from the methanolic extracts of eight soybean cultivars was measured according to Ramadan et al. [33] Briefly, 500 µL of a methanolic solution of phenolic extract at different concentrations (25,50. 100, 200, 400, and 800 µg/mL) was mixed with 2 mL of a freshly prepared methanolic solution of DPPH • (1 mM), Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Then, 1 mM methanolic solution of DPPH • without any addition was used as a control. The mixture was vortexed and incubated at room temperature for 30 min. The absorbance was read at 517 nm using a spectrophotometer. The antioxidant activity was calculated from the following Equation: Antioxidant activity (%) = (Ab control − Ab sample)/ Ab control × 100.

FRAP-Reducing Antioxidant Power
The FRAP reagent was prepared using a previously described method [34]. The stock solutions included 300 mM acetate buffer (3.1 g C 2 H 3 NaO 2 ·3H 2 O and 16 mL C 2 H 4 O 2 ), pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) the solution in 40 mM HCl, and 20 mM FeCl 3 ·6H 2 O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl 3 ·6H 2 O solution and was then warmed at 37 • C before usage. For the assay, 1 mL of extract solution was mixed with 1 mL of working FRAP reagent. Then, the mixture was kept at room temperature for 30 min. The absorbance of the reaction mixture was measured at 593 nm using a spectrophotometer. FRAP results were expressed as µmol Fe 2+ equivalents per g of extract using the calibration curve for FeSO 4 .
The cells were seeded in 96-well microplates at a density of 10 × 10 3 cells/well and were grown for 24 h at 37 • C in 5% CO 2 before the addition of the samples. The cells were treated with various concentrations (12.5, 25, 50, 100, and 200 µg/mL) of soybean cultivars methanolic extracts dissolved in phosphate-buffered saline (PBS). Cell viabilities were determined after 48 h incubation using the colorimetric MTT assay (Promega, Madison, WI, USA) [35]. The cell viability (%) was estimated based on the levels of formazan production, according to the absorbance at 550 nm. Triton X-100 (10 µL of a 10% solution) was used as the positive control, whereas untreated cells (0 µg/mL vehicle only) were used as the negative control. The percentage of cell viability was calculated by the following formula: cell viability (%) = (Ab sample/Ab control) × 100 Cytotoxic activity (%) of soybean extracts was calculated using the following formula: Cytotoxic activity (%) = 100 % − cell viability (%)

Statistical Analysis
The data of all studied characteristics of all soybean cultivars were statistically analyzed. In every trial, the order of samples was randomized and performed in three replicates per cultivar. Moreover, the experimental trials were conducted on different days. Analysis of variance (ANOVA) was performed for a completely randomized design on all data. The differences among cultivars, concentrations, and their interactions were determined by Tukey's range test (p ≤ 0.05).

Conclusions
Giza 35 exhibited the highest cytotoxic activity. In conclusion, Giza 35 cultivar recorded the highest TPC, TFC, antioxidant activity, and cytotoxic activity. Therefore, this cultivar can be used as a source for the production of pharmaceutical and medicinal products rather than as a nutritional source of protein.